Background: The problem of adulterated meat has become one of the greatest food safety issues in the world. It is reported that the meat used for adulteration includes fox meat, raccoon meat, mink meat, mouse meat, and so on. Although this kind of meat is edible in some areas, such meat is potentially harmful to human health because it is easy to carry bacteria, viruses, and harmful substances. The harm of mouse meat is most frightening. Therefore, it is urgent to develop a fast, accurate, and simple method to effectively identify mouse meat. Methods: In the present study, a new method of isothermal amplification based on the 16S ribosomal RNA gene of the mitochondrial DNA of the mouse was developed. The method is meant to improve the loop-mediated isothermal amplification (LAMP), separating the forward inner primers and backward inner primers, greatly reducing the nonspecific amplification of the method. Results: We have successfully obtained a set of best primers. The developed system allowed for the detection of 0.5% mouse meat from meat mixture effectively and specifically. The best ratio of the primers (F3: F2: F1: RF) was 1:2:2:8, and the optimum concentration of DNA template was 0.35 ng/μL. Conclusions: The assay has great specificity and sensitivity for detecting mouse meat and could provide specific positive results within 1 h. Highlights: We found a new approach of isothermal amplification to detect mouse source components. The LOD is determined to be 0.5 mg/mg. This new method is easy to perform and is able to provide rapid results in the specific detection of mouse meat sources.